1. Field of the Invention
The present invention is a method for immunoassays by using infrared reflection absorption spectroscopy. Essentially, the invention immobilizes protein molecules such as antigen or antibody on an infrared reflective substrate and detects the signals of the proteins by using the infrared reflection absorption spectroscopy. The present invention can be applied on the biosensor detection field.
2. Description of the Related Arts
Among the many types of biological assay techniques, immunoassay has been gaining favorable recognitions in recent years, because one positive attribute is that it uses proteins that each reacts quite exceptionally; hence, immunoassays have a higher level of sensitivity than other assay techniques. Essentially, immunoassays rely on general principles in immune reactions by taking an antibody and an antigen that was recognized mutually and combining them into a complex. This antibody-antigen complex is then used to determine the existence of antibody and antigen in a given medium.
When determining a method of detection that uses an antigen-antibody complex, scientists often rely on electrical and electrochemical, mass-sensitive, magnetic or optical, as well as other detecting means. Basically, electrical or electrochemical detecting means measure changes in electrode surface mass concentration caused by electron transfer reactions. Mass-sensitive detecting means interpret signals released by a change in mass. Magnetic detecting means use paramagnetic tracers to sense strength of magnetic field for determination of the concentration of the reactant. Optical detecting means begin with tagging a probe (such as biological fluorescent matter, chemical light emission material or dye) on the matter to be measured. When the matter and the probe have undergone biological reactions, the probe generates optical signals to be measured. Current optical detectors are primarily based on principles of light emission, surface plasmon resonance and evanescent wave absorption.
Principles of light emission, such as fluorescence or luminescence, are very well used because they can generate information that can be detected very quickly and directly. However, the immunoassays based on fluorescence generally require that a sandwich be formed by combining the antibody-antigen with a labeled antibody; thus this is not a single-step process. Furthermore, the fluorescence interference is often encountered in the UV-visible region due to naturally occurring or other contaminating fluorophores; this has led to major efforts to develop near-IR fluorophores for labeling the antibodies, since naturally occurring fluorescence is mostly confined to the UV-visible regions.
In the detection of surface plasmon resonance, the antibody molecule is immobilized on the metal surface such as gold or silver, which was previously deposited on the base of the optically transparent prism. Polarized light is incident with the appropriate resonance angle into the prism as to cause a resonance of light waves inside of the metal surface. The resonance angle would change when the media on the metal surface has a different refractive index. Such change can be used for assaying biomolecules. Although surface plasmon resonance is a very sensitive technique that allows for immediate detection of matters, it suffers from the fact non-specific absorption can occur on the immobilized surfaces, e.g., proteins can cling to the surface and cause a change in wavelength or angle of maximum resonance, and this would be falsely interpreted as the antigen.
Evanescent wave measuring means has been used in immunoassays in recent years. Such method of measurement is preferred for being direct and quick. Evanescent wave is generated on the interface of two light media. When the interfacing angle exceeds a critical angle, light wave will be total internal reflected from a heavy meson. Evanescent wave then is the electromagnetic wave that pierces through the heavy meson. However, the problem with evanescent wave measuring means is that there are very limited amount of parts for such device and that it can only measure one matter at a time, making measuring a large amount of matters very difficult.
There are many inconveniences in contemporary light spectroscopy, such as the difficulty in labeling light probes in fluorescence or luminescence techniques, or signal interference in the background. Thus, many have tried to develop new optical spectroscopy techniques. Among the new techniques being developed, infrared light has received a lot of attention as a new technique in light spectroscopy.
Infrared spectroscopy has contributed to protein structure analysis in the past and it is useful for the characterization of protein secondary structure and the identification of protein components. Since most biomolecules have specific IR spectra, it is possible to isolate structural effects of a protein interacting with substrates. This principle can be applied to bioassay in a biosensor. Infrared light allows experimenters to quickly obtain full light spectrum, and to easily prepare samples. Furthermore, infrared light can eliminate problems associated with the need for light probes in fluorescent light spectroscopy. Hence the present invention utilizes infrared spectroscopy to solve problems stated above.